Association of eosinophil‐derived neurotoxin levels with asthma control status in patients with aspirin‐exacerbated respiratory disease

Abstract Background The long‐term goals of asthma treatment are to achieve well control of symptoms and to minimize the future risk of asthma exacerbation. Identifying biomarkers for uncontrolled asthma is important for improving the asthma outcome. This study aimed to investigate the association of the levels of eosinophil‐derived neurotoxin (EDN) with asthma control status in specific asthma phenotype, aspirin‐exacerbated respiratory disease (AERD), and aspirin‐tolerant asthma (ATA). Methods A total of 136 adult asthmatics, including 47 asthmatics with AERD and 89 asthmatics with ATA, were enrolled. Plasma, sputum, and urine were collected at enrollment and the levels of EDN were measured by the K‐EDN ELISA kit. Urinary leukotriene E4 (LTE4) level was measured using liquid chromatography–mass spectrometry (LC‐MS)/MS methods. Asthma control status was evaluated according to the GINA guideline, asthma control test and asthma control questionnaire scores. Results In the total study subjects, sputum levels of EDN as well as of urine and plasma EDN showed significantly higher levels in patients with uncontrolled asthma than in those with well‐controlled or partly‐controlled asthma (ANOVA, p < 0.001); in patients with AERD, the sputum EDN levels showed significant correlations with ACT, ACQ, and AQLQ scores (p = 0.010, r = −0.536, p = 0.001, r = 0.665, and p < 0.001, r = −0.691, respectively), while no differences were noted in patients with ATA. Sputum EDN level was the only significant factor for ACT, ACQ, and AQLQ scores in patients with AERD (p = 0.001, p < 0.001, and p < 0.001, respectively) in the multivariate analysis adjusting for age, sex, peripheral eosinophil count, and urine LTE4. The ROC curve analysis demonstrated that sputum EDN can predict uncontrolled asthma with 80% sensitivity and 88.2% specificity for ACT ≤ 19 (area under the ROC curve [AUC] = 0.824, p = 0.019); 71.4% sensitivity and 86.7% specificity for ACQ ≥ 1.5 (AUC = 0.752, p = 0.049) only in AERD patients. Conclusion The level of sputum EDN may be a potential biomarker for identifying the asthma control status in patients with AERD.


| INTRODUCTION
Asthma is a heterogeneous disease which is characterized by chronic airway inflammation. 1 Asthma can be classified as that with and without T H 2 inflammation. 2 T H 2-high asthma endotype typically shows eosinophilic inflammation, whereas T H 2-low asthma endotype is associated with the neutrophilic or paucigranulocytic inflammation. 3 Eosinophilic inflammation induces airway remodeling and loss of asthma control, resulting in frequent asthma exacerbations (AE). 4,5 The long-term goals of asthma treatment are to achieve well control of symptoms and to minimize the future risk of AE. 1 Estimated annual frequencies of AEs per patient were reported to be 0.34-0.91 even in adult asthmatics having been treated with antiasthmatic medications. 6 In a large asthma cohort study in Korea, about 28.6% of asthmatics experienced ≥1 AE and 8.5% experienced ≥3 AE within the first 2 years of treatment. 7 Although the loss of asthma control is more frequent in patients with severe asthma (SA) than in those with mild to moderate asthma, it can occur regardless of asthma severity. 8 Uncontrolled asthma (UA) is a critical factor for asthma-associated morbidity and mortality and may increase health care cost of asthmatics and government public health agencies. 9 Therefore, identifying the biomarkers for UA is important for improving asthma outcome.
To date, several studies have shown that T H 2-driven biomarkers, including peripheral blood eosinophil count (PEC), sputum eosinophil count (SEC), and fractional exhaled nitric oxide (FeNO) could predict poor asthma outcome. 10,11 In clinical practice, SEC measurement requires specific technique with facility and sometimes shows variable results influenced by current treatment. Assessment of FeNO is not strongly recommended for outcome measurement due to the inconsistent study results. 12 Considering the consistency of the result and convenience of the measurement, PEC is the most frequently used biomarker; however, it may not always exactly reflect the degree of airway inflammation. 13 Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, chronic rhinosinusitis with nasal polyp, and hypersensitivity to nonsteroidal anti-inflammatory drugs (NSAIDs)/aspirin. AERD represents a distinct endotype with dysregulation of arachidonic acid metabolism and upregulated T H 2 inflammation. 14 Overproduction of cysteinyl leukotrienes which potentially induce eosinophilic inflammation is the hallmark of AERD in pathogenic mechanism. It has been reported that asthmatics with AERD are most likely to have severe disease, which has a higher risk of UA, SA, and AE. 15 Although urinary leukotriene E 4 has been suggested as a biomarker for the diagnosis of AERD, 16,17 biomarkers for UA in AERD patients are lacking.
In the present study, we prospectively enrolled asthmatics with AERD and ATA that showed various asthma control status to evaluate the association of the levels of eosinophil-derived neurotoxin (EDN) with clinical and inflammatory parameters as well as asthma control status.

| Study design and study population
A total of 136 patients with asthma were prospectively enrolled at Ajou University Hospital (Suwon, South Korea). Asthma was diagnosed according to the Global Initiative for Asthma guideline (GINA) 2022 by the allergy specialists. 1 Asthmatics were classified into three groups according to their symptom control status: UA, partlycontrolled asthma (PA), and well-controlled asthma (CA). Exclusion criteria for enrollment were as follows: (1) asthmatics who had been treated with biologics, including omalizumab, mepolizumab, reslizumab, and dupilumab within 130 days of enrollment; (2) current smokers or ex-smokers who quit smoking within 30 days of enrollment; and (3) asthmatics whose controller medications were changed within 7 days of enrollment.
AERD was defined by a typical clinical history (recurrent exacerbation of upper or lower respiratory reactions after ingestion of NSAIDs/aspirin) and/or a positive response to the lysine-aspirin bronchial provocation test (Lys-ASA BPT). The Lys-ASA BPT was performed with increasing doses of Lys-ASA solution up to 300 mg/ml using the method previously reported. 18 The Lys-ASA BPT result was considered positive if forced expiratory volume in one second (FEV 1 )% was decreased by more than 20% after the challenge. ATA was defined when subjects showed negative results to the Lys-ASA BPT or denied any upper or lower respiratory tract symptom changes after ingestion of NSAIDs/aspirin. Asthma control status was evaluated according to the GINA guideline, 1 asthma control test (ACT), and asthma control questionnaire (ACQ-6: ACQ mean of six individual item scores). 19 UA was defined when ACT ≤ 19 or ACQ ≥ 1.5. 1 SA was diagnosed according to the definition of international European respiratory society/American thoracic society guidelines. 20 Eosinophilic asthma was defined as the PEC ≥ 300/μl.

| Clinical data and sample collection
At the day of enrollment, peripheral venous blood, sputum, and urine samples were collected from the subjects between 8:00 a.m. and 11:00 a.m., when the patients had maintained on controller medications. Pulmonary function test, PC20 methacholine, FeNO measurement, and questionnaires survey using the ACT, ACQ-6 (ACQ mean of six individual item scores), and asthma quality of life (AQLQ[S]) were performed on the same day of enrollment.
Serum total immunoglobulin E level was measured using Immuno-CAP (ThermoFisher Scientific, Waltham, MA, USA). All subjects gave written informed consent at the time of enrollment, and the study was approved by the Institutional Review Board of Ajou University Hospital (AJIRB-BMR-SUR-15-498).

| Measurement of EDN and LTE 4
The samples of plasma, sputum, and urine were collected at enrollment and stored at −70°C. Levels of EDN in plasma, supernatant of sputum, and urine were measured using K-EDN kit (SKIMS-BIO Co., Seoul, Korea) as previously described. 21 Serum and urine levels of leukotriene E4 (LTE 4 ) were analyzed by liquid chromatographytandem mass spectrometry. LTE 4 -d5 (Cayman Chemical Company, Ann Arbor, MI, USA) was used as a deuterated internal standard.
Chromatographic separations were performed using the Waters Acquity UPLC system (Waters) with a Hypersil GOLD column  was used for the production of graphs.

| Clinical characteristics of the study subjects
A total of 47 patients with AERD and 89 patients with ATA were enrolled. Table 1 shows the demographic data from the study subjects. Patients with AERD showed higher PEC than those with ATA (p = 0.013). The number of atopic patients were higher in ATA patients (p = 0.004). There were no other significant differences between patients with AERD and ATA.

Levels of sputum, urine, and plasma EDN according to asthma control status defined by the GINA guideline
In the total number of study subjects, the sputum and urine levels of EDN showed significant difference among CA, PA, and UA groups defined by the GINA guideline (ANOVA, p = 0.002 and p = 0.049). The sputum and plasma levels of EDN were significantly higher in the UA group than in the others (p = 0.046 and p = 0.006). The urine and plasma levels of EDN were significantly lower in the CA group than in the others (p = 0.030 and p = 0.041) ( Figure 1).
In patients with AERD, sputum levels of EDN showed significant difference among the CA, PA, and UA groups (ANOVA, p < 0.001), while no differences were noted in the urine and plasma levels of EDN. The levels of sputum EDN were significantly higher in AERD patients with UA than in those with CA or PA (p < 0.001 for all). Patients with ATA showed no differences in the levels of EDN according to asthma control status by the GINA guideline.

| Correlation between EDN levels and asthma control questionnaire scores
The sputum levels of EDN were significantly correlated with the ACQ and AQLQ scores in asthmatic subjects (p = 0.007, r = 0.369, and

| Correlation between EDN levels and pulmonary function tests
The sputum EDN levels were negatively correlated with the predicted % value of FEV 1 or FEV 1 /FVC (forced vital capacity) ratio in asthmatic subjects, especially in patients with AERD (p = 0.001, r = −0.646 and p = 0.008, r = −0.551, respectively), but not in those with ATA. In addition, urine, and plasma levels of EDN were significantly correlated with FEV 1 /FVC ratio in asthmatic subjects. However, ATA patients had no correlations between the levels of urine/ plasma EDN and pulmonary function test results (Table 2).

| Correlations between EDN levels and inflammatory biomarkers
The EDN levels showed positive correlations with PEC (sputum EDN:  Table 3 shows the correlation of the LTE 4 and EDN levels in asthmatics with AERD and ATA patients.

| Factors associated with asthma control status
In the univariate analysis, PEC and sputum EDN level were significant   interleukin-25, and interleukin-33, activate mast cells and eosinophils and then leads to release cysteinyl leukotrienes. 27,28 Urinary LTE 4 has been extensively studied as a reproducible biomarker determining the phenotype of AERD and reported to be elevated in patients with uncontrolled AERD. 29,30 Elevated levels of PEC or FeNO are also known for poor asthma control and risk factor for AE. 1,11,31 The present study demonstrated that EDN levels (plasma, urine, and sputum) are the most important factor predicting UA in patients with AERD, but not in those with ATA. EDN was significantly correlated with PEC and LTE 4 . In the multivariate analysis sputum EDN remained as the only significant factor for poor asthma control in AERD patients. These results were consistently found, regardless of the outcome parameter used for the assessment of asthma control status including ACT, ACQ, or AQLQ scores. Several kinds of specimens have been used to analyze the levels of eosinophil degranulation products in asthma; however, there is no definite consensus on which sample is the most suitable to predict asthma control status. In the present study, EDN levels in sputum samples showed better predictability for UA, suggesting that EDN levels found in airway secretions (target tissues of asthma) may better predict airway inflammation and clinical outcome (asthma control status). The potential clinical implication of this observation warrants further studies.
Meanwhile, we could not find statistical significance of PEC, SEC, or FeNO levels for predicting poor asthma control in the multivariate analysis. This is in agreement with the previous studies that showed better performance of EDN level to assess asthma control status than PEC. 24,26 These findings collectively suggest that eosinophil activity may be a pivotal factor inducing poor asthma control rather than the level of PEC itself in patients with AERD.
As AERD is not fully understood by enhanced production of type 2 cytokines or cysteinyl leukotrienes, novel molecules related to activated eosinophils have been proposed to be implicated in AERD pathogenesis. 32 Eosinophil extracellular trap (EET) which is released from activated eosinophils contains a mixture of web-like DNA fibers and granule proteins. 33 EET-forming eosinophil counts were positively correlated with serum EDN levels and negatively correlated with FEV 1 % in patients with SA. 34 In this context, EET and EDN have been postulated to be involved in the mechanism of activated eosinophils and potential therapeutic target for AERD. 32  showed significantly reduced levels of EPO in bronchoalveolar lavage. 37 These studies have shown that eosinophil degranulation processes are critical for the progression and severity of type 2 airway inflammation. Consistent with this experimental study data, the present study also demonstrated that clinical outcome (asthma control status) was more closely related with the amount of degranulated product (EDN) than with eosinophil count itself. Therefore, the level of eosinophil degranulation product (EDN) may be a useful biomarker for predicting asthma control status in patients with AERD, even in patients on maintenance medication.
In addition, specific polymorphisms in the RAB1A gene were associated with the risk of AERD and a greater decline in FEV 1 % after aspirin challenges. 38 A recent transcriptomic study also identi-